The complete chloroplast genome and phylogentic results support the species position of Swertia banzragczii and Swertia marginata (Gentianaceae) in Mongolia

Background Swertia banzragczii and S. marginata are important medicinal species in Mongolia. However, their taxonomic positions and genetic backgrounds remain unknown. In this study, we explored the complete chloroplast genomes and DNA barcoding of these species and compared them with those of closely related species within the subgenus to determine their taxonomic positions and phylogenetic relationships. Result The chloroplast genomes of S. banzragczii and S. marginata encoded 114 genes, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Among them, 16 genes contained a single intron, and 2 genes had two introns. Closely related species had a conserved genome structure and gene content. Only differences in genome length were noticed, which were caused by the expansion and contraction of the inverted repeat (IR) region and loss of exons in some genes. The trnH-GUG–psbA and trnD-GUC–trnY-GUA intergenic regions had high genetic diversity within Swertia plastomes. Overall, S. banzragczii and S. marginata are true species and belong to the subgenus Swertia. Conclusions These results provide valuable genetic and morphological information on rare and subendemic Swertia species in Mongolia, which can be used for further advanced studies on the Swertia genus.


Background
Swertia L. is one of the largest genera in the Gentianaceae Juss.and comprises 160 species (Ho and Liu 2015;Nampy et al. 2015;POWO 2023).Members of the Swertia genus are mainly distributed in the temperate and alpine regions of Asia, Europe, and the Americas (Ho and Liu 2015).This genus has great medicinal importance and is the most taxonomically challenging taxon in the family, largely because of the morphological similarities of species within this genus and closely related genera (Chassot et al. 2001;Ho and Liu 2015;Cao et al. 2022).It is distinguished by a rotated corolla and the presence of coralline nectariferous glands in related genera within the family (Ho and Liu 2015).Furthermore, the nature and shape of these glands are two of the main diagnostic characteristics for species delimitation in this genus (Favre et al. 2015).
Four to five species of Swertia have been recorded in Mongolia, i.e., Swertia banzragczii Sanchir, S. marginata Schrenk (or S. komarovii Pissajuk.),S. obtusa Ledeb.and S. dichotoma L. (Baasanmunkh et al. 2022a).The current taxonomic status of S. banzragczii and S. marginata (or S. komarovii Pissajuk.) is still doubtful (Ho and Liu 2015).However, these species are currently accepted taxa on websites such as the Global Biodiversity Information Facility (GBIF) and Plants of the World Online (POWO 2023).The two Swertia species have not been fully investigated based on especially molecular evidence, including DNA barcoding and plastomes.
The development of barcoding markers has greatly advanced our understanding of the relationships between morphologically similar species.Nuclear ribosomal DNA internal transcribed spacer (ITS) and matK, trnL, trnL-F, and trnS of chloroplast markers mainly used in the phylogenetic study of Swertia; however, it has been revealed that Swertia is strongly paraphyletic to other genera within the Gentianaceae family (Chassot et al. 2001;Xi et al. 2014).Therefore, more genomic studies are needed for reconstructing the phylogenetic relationships of Swertia.Recently, the chloroplast genome has been widely used in plant system evolution, related species identification, genetic diversity analysis, chloroplast genetic engineering, and other applications due to the development of high-throughput sequencing technology (Yang et al. 2016;Li et al. 2021;Fang et al. 2023;Nyamgerel et al. 2023Nyamgerel et al. , 2024)).The chloroplast genome (plastome) has a conserved structure, encodes 120-130 genes, most of which encode part of the photosynthetic apparatus of the organelle (Jensen and Leister 2014), and is maternally inherited (Wicke et al. 2011).Therefore, plastomes have stronger species-discriminating abilities and provide more information on genetic variation than ultra-barcodes (Hollingsworth et al. 2016).Several studies have explored the complete chloroplast genome of Swertia species, mainly from China (Bi et al. 2020;Du et al. 2022;Yang et al. 2020Yang et al. , 2022Yang et al. , 2023;;Cao et al. 2022).These studies determined the plastome structure, genetic variety, and phylogenetic relationships across 26 species of Swertia and compared them with those of the subtribe Swertiinae species.In addition, the ITS marker is widely used for phylogenetic studies and ITS region of many Swertia species was sequenced.The ITS is biparentally inherited and has a higher mutation rate than maternally inherited plastome (Wolfe et al. 1987).However, little is known about the comparative plastome analysis with phylogenetic relationships of Swerita species in Mongolia.To address this gap, therefore, we investigated the plastome analysis and DNA barcoding on previously poorly known two Mongolian Swertia species.
In this study, we pursued the following aims: (i) to explore the complete chloroplast genomes of S. banzragczii and S. marginata; (ii) to identify the candidate regions from chloroplast genome to use as barcoding markers; (iii) compared chloroplast genome and barcoding region to determine the taxonomic positions and phylogenetic relationships of the closely related species.

The chloroplast genomes of S. banzragczii and S. marginata
The chloroplast genomes of S. banzragczii and S. marginata were sequenced for the first time.In total, 54,848,640 and 66,783,272 reads were produced for S. banzragczii and S. marginata, respectively (Table 1).After de novo assembly, a single contig was generated for each plastome.The two plastomes had typical quadripartite natural structures comprising a large single copy (LSC), a pair of inverted repeats (IR), and a small single copy (SSC) (Fig. 1).The total length of S. marginata plastome was shorter than the S. banzragczii plastome in all regions (Table 1).The overall guanine-cytosine (GC) content of the two plastomes was similar, differing by 0.1% in total length.The two plastomes encoded 114 genes, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes.Among them, 18 genes (7 protein-coding, 7 tRNA, and 4 rRNA genes) were duplicated in the IR regions.Sixteen genes contained a single intron, and two genes had two introns (Table 2).Of the 18 genes, 12 intron-containing genes (trnK-UUU, rps16, trnG-UCC, atpF, rpoC1, trnL-UAA, trnV-UAC, petB, petD, and rpl16) were located in the LSC and 1 gene (ndhA) was located in the SSC.Five genes (rpl2, ndhB, rps12, trnI-GAU, and trnA-UGC) were duplicated in the IRs.The rps12 gene was a trans-spliced gene with 5′ end located in the LSC and 3′ end located in the IR.

Chloroplast genome comparison of genus Swertia species
The cp. genome size, structure, and gene positions of S. banzragczii and S. marginata were compared with those of five closely related species within the subgenus Swertia: S. bifolia Batalin, S. erythrosticta Maxim., S. souliei Burkill, S. przewalskii Pissjauk, and S. wolfgangiana Grüning.The gene content and location in all regions of closely related species were similar, and the plastomes did not display any species-specific inversion or translocation (Fig. 2).The IR of S. banzragczii and S. marginata were 25,855 and 25,755 bp, respectively.The borders between the IR regions and two single-copy regions (LSC and SSC) were similar to closely related species (Fig. 3).The rps19, ndhF, and ycf1 genes spanned the LSC/IRb, IRb/SSC, and SSC/IRa junctions, respectively.However, the ndhF gene of S. marginata was 935 bp shorter than that of the others and was entirely located in the SSC region.Relative synonymous codon usage (RSCU) was computed for protein-coding genes.These genes revealed 26,770 and 26,596 codons in S. banzragczii and S. marginata plastomes, respectively, and they had similar patterns in both plastomes, with the highest RSCU values for the AGA (1.9) codon of arginine (Arg) and the highest coding rate for leucine (Leu; 5,216 codons), isoleucine (Ile; 4,138 codons), and phenylalanine (Phe; 3,603 codons) (Fig. 4).Codons with A or T at the third position had a strong codon bias.The two species studied exhibited similar patterns.All coding sequences (CDS) contained a standard ATG start codon.Among the three stop codons, TAA was the most common.The sequence identities of the seven Swertia species were analyzed using mVISTA, Transfer RNA genes (tRNA)  psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT,   As expected, the genic regions were more conserved than the intergenic regions, and genes in the IR regions were highly conserved, followed by those in the LSC and SSC regions (Fig. 5).
We analyzed the genetic divergence of genes and intergenic regions in the Swertia genus.Overall, Swertia plastomes had an average genetic diversity (Pi) value of 0.17.The highest Pi values were observed in the trnH-UUU-psbA (0.082) and trnD-GUC-trnY-GUA (0.016) intergenic regions of the LSC regions (Fig. 6).Among these genes, the highest value (0.67) was found for rps16 in the LSC region.The genic and intergenic regions in the IR region were relatively constant compared to those in the LSC and SSC regions.Common chloroplast barcoding markers such as matK, rbcL, trnL-F, and trnH-psbA showed relatively low genetic diversity within the Swertia genus.

Phylogenetic position of S. banzragczii and S. marginata
The alignment of Swertia plastomes consisted of 152,075 bp, of which 82,024 bp (94.2%) were similar, and 5,051 bp (5.8%) were parsimony-informative.The alignment of the ITS sequences consisted of 622 bp, 153 of which were parsimony-informative.The topologies of maximum parsimony (MP) and Bayesian interference (BI) were highly congruent with the datasets and supported by strong bootstrap values and posterior probabilities.Swertia species were divided into two clades (I and II) based on the plastome and the ITS trees (Fig. 7).The clade I consist S. banzragczii and S. marginata with section Swertia including S. bifolia, S. erythrosticta, S. souliei, S. przewalskii and S. wolfgangiana (Fig. 7).Swertia perennis was used only in ITS tree and formed monophyletic group with S. banzragczii.The clade II include species from section Swertopsis and Platynema.However, the relationship of species within the subbranches produced incongruent topologies in the plastome and ITS tree.

Discussion
The complete plastome of Swertia has recently been studied (Bi et al. 2020;Yang et al. 2020;Cao et al. 2022;Qing et al. 2022;Yang et al. 2022;Yang et al. 2023).However, previous studies have not explored Mongolian Swertia species.In this study, we sequenced and characterized the plastomes and determined their taxonomic positions based on the genetic and morphological features of two Swertia species in Mongolia.
The complete plastomes of S. banzragczii and S. marginata have a typical quadripartite structure with one LSC, one SSC, and two IR regions.They have relatively conserved features, genes, and GC content of sequences, similar to most species in Gentianaceae, and no significant structural rearrangements (Fig. 2) (Zhang et al. 2020;Yang et al. 2023).The Swertia plastome ranges from 149,089 bp (S. cincta) to 153,872 bp (S. banzragczii), and the difference in length is caused by expansion and contraction of the IR region, as previously reported in the Swertia genus (Cao et al. 2022;Yang et al. 2022).The IR lengths of Swertia ranged from 25,331 to 25,890 bp, and rps19 and ycf1 spanned the LSC/IRb and SSC/IRa junctions, respectively, within all species.The IRb/SSC junction varied greatly owing to the extension of 5-100 bp of ndhF from SSC to IRb.In the S. banzragczii plastome, the IRb/SSC junction includes ndhF, which consists of 54 and 2,175 bp in the IRb and SSC regions, respectively (Fig. 3).In contrast, the IRb/SSC junction of S. marginata plastome did not contain ndhF, which was located entirely in the SSC region.Synonymous codons are genetic codons encoding the same amino acids.Codon usage bias describes the frequency of synonymous codons that vary among different species.Codon usage patterns can be used to evaluate genetic evolution, environmental adaptation, and evolutionary relationships between species (Athey et al. 2017).Previous studies have shown that analysis of codon bias in the Swertia plastome revealed that leucine, isoleucine, and phenylalanine had the highest coding rates (Yang et al. 2022), and similar results have been reported in S. banzragczii and S. marginata plastomes (Fig. 4).In addition, the plastomes of the subtribe Swertiinae had a weak codon bias, which might be related to the conserved of plastome (Yang et al. 2023).
The plastome has a slower rate of nucleotide substitution in the coding region and a more conservative gene structure and content than the nuclear genome.It can act as a molecular marker for the interpretation of evolutionary relationships and improve phylogenetic resolution at lower taxonomic levels (Wicke et al. 2011).The sequence divergence of 23 Swertia species was discovered by Yang et al. (2022); five (rpoC1, ccsA, ndhI, ndhA, and rps15) protein-coding genes and five (trnH-psbA, psaA-ycf3, cemA-petA, ycf15-trnL, and ccsA-ndhD) non-coding regions had the highest variability in the plastome.In addition, the plastomes of 26 Swertia species were used in this study to calculate genetic diversity; the rps16 gene and certain intergenic regions (trnH-psbA, trnD-trnY-, ndhF-rpl32, trnK-rps16, ccsA and ndhD) showed the highest values (Fig. 5).Among these regiosn, trnH-psbA used to barcoding marker in previous phylogenetic studies of Swertia (Groff et al. 2015).Furthermore, Yang et al. (2022) reported that some species of Swertia have lost an exon of rps16, which has been found in several Gentianaceae species, and that its structure was similar in all species.
The phylogenetics of Swertia have been the subject of extensive research.Previous phylogenetic studies of Swertia species have used nrDNA (ITS) and plastid (atpB-rbcL, matK, trnL, trnL-trnF, trnS-ycf9, and trnH-psbA) barcoding markers (Chassot et al. 2001;Xi et al. 2014;Groff et al. 2015).These findings indicate that Swertia is paraphyletic, with certain species sharing a foundational polytomy with other closely related genera.Moreover, pollen morphology analysis revealed that the Swertia genus has 14 different lineages at the respective phylogenetic positions within the Swertiinae subtribe (Chassot and Hagen 2008).Recently, the taxonomic relationships of Swertia have been revised using the complete chloroplast genome (Cao et al. 2022;Favre et al. 2010;Yang et al. 2022;Yang et al. 2023).Some studies have investigated the connections between Swertia and other genera in Gentianaceae, such as Gentianopsis, Halenia, Lomatogonium, Lomatogoniopsis, and Sinoswertia (Park et al. 2019;Li et al. 2021;Yang et al. 2023).These findings support the polyphyly of Swertia, which is consistent with previous molecular investigations but provides significantly better resolution.In this study, we first determined the phylogenetic position of S. banzragczii and S. marginata, which were formed the monophyletic clade with section Swertia species based on the plastome and nrDNA (ITS) phylogenetic trees (Fig. 7B).In previous studies, the section Swertia always formed a well-supported monophyletic clade, whereas the Swertia genus was polyphyletic in phylogenetic trees (Xi et al. 2014;Yang et al. 2022).The phylogenetic positions of the species within Swertia were inconsistent among the phylogenetic tree of plastomes and nrDNAs, specifically relationships of species within clade II.This incongruence was likely the result of different inherited backgrounds and mutation rates of ITS and plastome (Wolfe et al. 1987).More Swertia species need to be sequenced in further studies to better understand their phylogenetic relationships.

Swertia banzragczii
Taxonomic notes.Swertia banzragczii was first described in Western Mongolia (Sanchir 1984).Later, this species was found in Xinjiang, China (Chen, 2011); however, its taxonomic status of S. banzragczii is uncertain (Ho and Liu 2015).Based on molecular evidence, our results showed that S. banzragczii is a true species that its current Altai endemic species has a narrow distribution in the Altai Mountain range (Erst et al. 2022).
Penology.Flowering and fruiting in July to September.General distribution and habitat.China (Xinjiang) and Mongolia (the Khovd and Mongolian Altai regions) (Baasanmunkh et al. 2022a).This species grows in alpine meadows, dwarf birch groves, sparse larch groves, and the borders of the alpine belt (Baasanmunkh et al. 2022b).
Conservation status.Swertia banzragczii was assessed as near-threatened at the global level, according to our previous study (Erst et al. 2023).

Swertia marginata
Schrenk, Bull.Sc.Acad.Petersb. x. 353 (1842).(Fig. 9) Synonym.Swerita komarovii Pissajuk.TYPE: Montes Taxonomic notes.The taxonomic status of S. marginata has been thoroughly addressed by Ho and Liu (2015).According to Ho and Liu (2015), S. komarovii Pissjauk.was treated as a synonym of S. marginata based on its morphological characteristics, but it is currently accepted as a species in GBIF (https://www.gbif.org/species/5596000).Swertia komarovii was first described in Russia and may have occurred in the Khusgul region of Mongolia (Pissjaukova 1961).Based on our ITS phylogenetic results (Fig. 7B), the Mongolian samples clustered with S. marginata; therefore, we confirmed that S. komarovii is a synonym of S. marginata.
Penology.Flowering and fruiting in July to September.General distribution and habitat.Afghanistan, China, Kazakhstan, Kyrgyzstan, Mongolia (Khuvsgul and Mongolian Altai regions), Russia, and Tajikistan (Ho and Liu 2015).In Mongolia, this species grows in wet meadows, syrts (elevated watersheds), along the banks of brooks, moraines, shrubs, rubbly sites, slopes, taluses, and rocks in spruce forests.
Conservation status.The conservation status is currently not assessed at the global level.

Conclusion
In this study, the plastomes of S. banzragczii and S. marginata from Mongolia were sequenced and assembled for the first time.The genome size, organization, and gene content were similar to those of other Swertia species.However, they varied slightly with respect to genome length and sequence of intergenic regions.Based on the phylogenetic analyses, they belonged to the subgenus Swertia.In general, our results demonstrated that S. banzragczii and S. marginata are true species and contribute more information towards resolving taxonomic ambiguities around Swertia species in Mongolia.Furthermore, this study used molecular evolution and phylogenetic analyses to determine the genetic connections within Swertia.

Plant material, DNA extraction, and sequencing
Fresh leaves of S. banzragczii and S. marginata were collected from Bayan-Ulgii and Khuvsgul provinces, Mongolia, and voucher specimens (UBU0005243 and UBU0030443) were deposited in the herbarium of the National University of Mongolia, Mongolia.Photographs of each species taken in the wild were obtained from field surveys conducted by the authors.Total genomic DNA was extracted from the silica gel-dried leaf material using the CTAB method (Doyle and Doyle 1987).DNA purity and concentration were quantified using agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer, respectively.
The nuclear ITS region (White et al. 1990) was used for amplification and sequencing.The PCR cycling parameters were 5 min at 94 °C, followed by 30 cycles of 45 s at 94 °C, 45 s at 56 °C, and a final 7 min at 72 °C.The PCR reaction (20 µL) includes 10X PCR buffer, 1.5 U Taq DNA polymerase, 0.2 mM dNTPs, 1 µM of each primer, and 100 ng genomic DNA.PCR products were subjected to gel electrophoresis and cleaned using a PCR clean-up kit.The purified PCR products were directly sequenced in two directions using the corresponding primers.Sequences were aligned using ClustalW (Thompson et al. 2003), and default settings and manual adjustments were made using BioEdit (Hall et al. 2011).Ambiguous nucleotide bases were corrected using the corresponding bases of sequences obtained using the reverse primer.The DNA sequences generated in this study have been deposited in GenBank (https://www.ncbi.nlm.nih.gov/).
The complete cp.genome library was constructed using the TrueSeq DNA Nano Kit protocol on the NectSeq 500 platform (Illumina, San Diego, CA, USA), following the extraction of total DNA.Trimmomatic software version 0.36 (Bolger et al. 2014) was used to eliminate adapter sequences and low-quality reads to mitigate biases in the analysis.A FastQC v. 0.11 (Antil et al. 2023) was used to

Genome assembly and annotation
De novoassembly was conducted using various k-mers with NOVOplasty (Nicolas et al. 2017), using the default settings.A BLAST analysis was conducted following the assembly of the complete plastome or draft plastome to determine the species with which each contig (or scaffold) exhibited similarity.S. banzragczii and S. marginata plastomes were annotated using the online software GeSeqAnnotation of Organellar Genomes (Tillich et al. 2017).After initial annotation, the tRNA genes were further checked using tRNAscan-SE v1.21 (Lowe and Chan 2016).We manually calibrated the start and stop codons of coding sequences in the software Geneious Prime 2023.2.1 (https://www.geneious.com).The gene map was drawn using Organ-ellarGenomeDRAW (OGDRAW) (Lohse et al. 2007).The newly sequenced plastomes were deposited in GenBank under accession numbers OR643855 (S. banzragczii) and OR786442 (S. marginata).

Phylogenetic analysis
The nrDNA (ITS) and plastome sequences were used to determine the phylogenetic positions of S. banzragczii and S. marginata within the Swertia genus.We constructed plastome datasets for 26 Swertia species using Gentianopsis paludosa (Hook.f.) Ma as an outgroup.The ITS dataset was constructed using 25 ITS sequences and Gentiana straminea Maxim.as an outgroup.Additional sequences of the compared species were downloaded from GenBank, and the accession numbers are listed in Table 3. Plastome alignment datasets were filtered to remove ambiguously aligned regions using GBlock ver.0.91.1 (Talavera and Castresana 2007).The best-fitting model for nucleotide substitutions was determined using the Akaike information criterion in jModelTest v2.1.10 (Darriba et al. 2012).Phylogenetic trees were constructed using the MP and BI methods.The MP analysis was performed using PAUP*v4.3.99.169.0 (Swofford and Documentation 1989).The branch-swapping algorithm for MP analysis was performed using tree bisection reconnection.The robustness of the tree was evaluated using 1000 bootstrap replication indices, and the consistency, retention, and composite indices were calculated.BI analysis was carried out using the MrBayes 3.2.6 (Ronquist et al. 2012) with two independent runs of four simultaneous chains executed for 5,000,000 generations using the Markov chain Monte Carlo algorithm.Trees were sampled every 5000 generations, and the first 25% were discarded as burn-in.The reconstructed trees were visualized using FigTree v.1.4.4 (Rambaut 2012).

Fig. 2
Fig. 2 Comparison of complete plastomes from seven Swertia species using the MAUVE alignment.Blocks in the top of each row are in the same orientation, while blocks on the bottom of each row are in the inverse orientation.Boxes under each genome map represent protein-coding genes (white), rRNAs (red), and tRNAs (green).Red blocks in the lowest bottom row are inverted repeat (IR) regions

Fig. 4
Fig. 4 Codon usage for 20 amino acids in the protein-coding genes of S. banzragczii and S. marginata.The colors in the bars are the same as that of the codons below the figure.RSCU: relative synonymous codon usage

Fig. 5
Fig. 5 Comparison of complete plastomes from seven Swertia species, including S. banzragczii as a reference, using the mVISTA software.The gray arrows above the alignment indicate genes.Different colors represent different regions (coding and non-coding).The horizontal axis indicates the coordinates within the plastomes.The vertical scale represents the percentage of identity, ranging from 50 to 100%

Fig. 6
Fig. 6 Comparison of the nucleotide diversity (Pi) values among the Swertia species.The mean Pi value is indicated by the blue line

Fig. 8
Fig. 8 Swertia banzragczii in Mongolia: A general habitat; B inflorescence; C flower front view; D flower; E cauline leaves

Fig. 9
Fig. 9 Swertia marginata in Mongolia: A general habitat; B flowers front view; C, D calyx; E cauline leaves

Table 1
Raw reads and genome assembly information for S. banzragczii and S. marginata plastomes

Table 2
Gene category of S. banzragczii and S. marginata plastomes